The overall objective of this project is to elucidate the molecular mechanisms involved in the regulation of the growth and function of adrenocortical cells. During the past year, we succeeded for the first time in detecting and characterizing the physiologically relevant receptors for ACTH on rat adrenocortical cells. We also prepared a well-characterized photoreactive derivative of the hormone and used it to identify the ACTH receptor on adrenocortical cells. In addition, the photoreactive peptide was employed to identify the site of interaction between ACTH and serum albumin, which may be physiologically important in the transport of the hormone in incubation. The role of calcium in the actions of ACTH was investigated by direct binding studies and photoaffinity labeling. The results clearly demonstrate that calcium is essential for the binding of ACTH to its receptor and for continued occupancy of the receptor. Extracellular calcium is not necessary for activation of steroidogenesis once the hormone is bound to the receptor and maintained on the receptor by covalent attachment. ACTH receptors in human adrenocortical cells were also characterized. A single class of binding sites with an apparent KD of 1.6 + 0.14 nM and a binding capacity of 3560 sites/cell was found. Concentration-response curves for cAMP and cortisol production were shifted to the left, with concentrations of ACTH required for half-maximal stimulation being 20-\and 800-fold less, respectively, than that for binding. (C)